DISTINCT ROLES OF C2A AND C2B DOMAINS OF SYNAPTOTAGMIN IN THE REGULATION OF EXOCYTOSIS IN ADRENAL CHROMAFFIN CELLS

Synaptotagmin that contains two repeats of C2 regulatory domains is co nsidered to be involved in neurotransmitter release. To reveal the rol es of synaptotagmin in the regulation of exocytosis, we examined the e ffects of antibodies against C2A and C2B domains on Ca2+-evoked catech olamine (CA) release from digitonin-permeabilized adrenal chromaffin c ells, resolving the Ca2+-evoked release into ATP-dependent priming and ATP-independent Ca2+-triggered steps. Anti-C2A antibody clearly reduc ed the ATP-independent release, suggesting that the C2A domain directl y facilitate or promote Ca2+-triggered step, vesicular fusion. In cont rast, anti-C2B antibody did not affect Ca2+-evoked release by itself, but significantly increased the spontaneous Ca2+-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C 2B domain inhibited both the ATP-independent Ca2+-evoked release and t he spontaneous release in a dose-dependent manner. The inhibition by I HPS was totally reversed by anti-C2B antibody and significantly revers ed by high concentration of Ca2+, These results suggest that IHPS bind ing to C2B domain arrests membrane fusion by presumably preventing int eraction of synaptotagmin with phospholipids or with proteins of plasm a membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of i ntracellular Ca2+. Binding of the increased intracellular Ca2+ to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.